The two aims of this proposal are focused on scavenger receptor B1 (SR-BI) and apolipoprotein Al (apoA-I) and their function in the lipoprotein cholesteryl ester (CE) selective uptake pathway. Previous studies with mice and rats and cultured human cells indicate that this pathway plays a major role in the uptake of high density lipoprotein (HDL) CE into the liver and steroidogenic cells. Studies in gene knockout mice show that SR-BI is the receptor responsible for HDL CE selective uptake and that apoA-I is the key HDL ligand for SR-BI. Recent studies indicate that SR-BI has multiple effects on cellular cholesterol metabolism including changes in plasma membrane properties. Aim 1 has four goals in which the mechanisms by which SR-BI alters plasma membrane properties will be investigated. Goal 1 will test the hypothesis that SR-BI is necessary for the formation of microvillar channels and the cell surface localization of HDL particles on steroidogenic cells. These experiments will compare wild type and SR-BI-deficient mice and will use ultrastructural analysis at the electron microscope (EM) level and HDL localization analyses at the light microscopic (LM) and EM levels. Goal 2 will test the role of caveolin-1 in SR-BI-mediated HDL CE selective uptake by comparisons of wild type and caveolin-1-deficient mice. These experiments include morphological analyses and in vivo analysis of HDL CE selective uptake. Goal 3 will test the hypothesis that SR-BI-induced alterations in plasma membrane morphology reflect changes in membrane phospholipids. Tandem mass spectrometry will be used to determine the phospholipid distribution and acyl tail compositions in membranes prepared from two cell types in which SR-BI alters membrane morphology, the mouse adrenal z. fasciculata cell and the insect Sf9 cell. Goal 4 will test the hypothesis the SR-BI is assembled in the plasma membrane of adrenal cells as a homo-oligomeric complex. Aim 2 is focused on the key issue of how SR-BI recognizes its ligand during docking of the HDL particle. These experiments use single cys mutants of apoA-I to provide site-specific chemical cross-linking to SR-BI. Sites of cross-linking in SR-BI will be determined by isolation of SR-BI peptides and identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry. These experiments test the hypothesis that SR-BI recognizes apoA-I via its amphipathic alpha-helical repeat units. Additionally, sites of cross-linking in SR-BI will identify key residues and receptor domains involved in the recognition of HDL particles. These studies will provide new and important information relevant to the role of HDL in protecting against atherosclerotic disease.